Activation of the β myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/NFATc1 pathway

Calcium is a key element in intracellular signaling in skeletal muscle. Changes in intracellular calcium levels are thought to mediate the fast-to-slow transformation of muscle fiber type. One factor implicated in gene regulation in adult muscle is the nuclear factor of activated T-cells (NFAT) isoform c 1, whose dephosphorylation by the calcium/calmodulin-dependent phosphatase calcineurin facilitates its nuclear translocation. Here, we report that differentiated C2C12 myotubes predominantly expressing fast-type MyHCII protein undergo fast-to-slow transformation following calcium-ionophore treatment, with several transcription factors and a transcriptional coactivator acting in concert to upregulate the slow myosin heavy chain (MyHC) beta promoter. Transient transfection assays demonstrated that the calcineurin/NFATcI signaling pathway is essential for MyHC beta promoter activation during transformation of C2C12 myotubes but is not sufficient for complete fast MyHCIId/x promoter inhibition. Along with NFATc 1, myocyte enhancer factor-2D (MEF-2D) and the myogenic transcription factor MyoD transactivated the MyHCP promoter in calcium-ionophore-treated myotubes in a calcineurin-dependent manner. To elucidate the mechanism involved in regulating MyHCO gene expression, we analyzed the -2.4-kb MyHCO promoter construct for cis-regulatory elements. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitation assays (ChIP), and nuclear complex coimmunoprecipitation (NCcolP) assays, we demonstrated calcium-ionophore-induced binding of NFATc I to a NFAT consensus site adjacert to a MyoD-binding E-box. At their respective binding sites, both NFATcI and MyoD recruited the transcriptional coactivator p300, and in turn, MEF-2D bound to the MyoD complex. The calcium-ionophore-incluced effects on the MyHC beta promoter were shown to be calcineurin-dependent. Together, our findings demonstrate calcium-ionophore-induced activation of the beta MyHC promoter by NFATc1, MyoD, MEF-21D, and p300 in a calcineurin-dependent manner.